20.109(S15):AIV detection assay and analysis (Day7)

Introduction

Real-time reverse transcription-polymerase chain reaction (RRT-PCR) allows researchers to monitor the results of PCR as amplification is occurring (this technique is also referred to as quantitative or real-time PCR). During RRT-PCR data are collected throughout the amplification process using a fluorescent dye. The fluorescent dye is highly specific for double-stranded DNA and when bound to DNA molecules the fluorescence intensity increases proportionate to the increase in double-stranded product. In contrast, the data for traditional PCR are simply observed as a band on a gel (remember back to M1D5).

The fluorescent dye binds to double-stranded DNA during the cycles of PCR. At the annealing temperature the primer (blue arrow) binds to the template (black line). During an incubation at the extension temperature the new copy of DNA (orange dashed arrow) is sythesized by the polymerase enzyme. The inactive fluorescent dye molecules present in the reaction (grey stars) bind to the newly generated double-stranded DNA and become activated (green stars). To eliminate clutter, the basepairs between the DNA strands were omitted. An animation of this process is linked here.

For the purpose of your AIV screening assay, your RRT-PCR data will be used to test the sensitivity of the primers you designed on Day 2 of this module. Primers with high sensitivity can better detect AIV from flu-positive bird samples. Why are primers with high sensitivity better? The sensitivity of the primer determines its ability to identify and bind the target sequence. If primer binding is enhanced then amplification is improved and the presence of AIV is more likely to be detected. In addition, primers with lower sensitivity can give false positives and hinder the development of models that aid researchers in understanding AIV abundance and epizootiology in wild birds.

Example amplification curve. These data were collected by Sp14 20.109ers!

Example melt curve. These data were collected by Sp14 20.109ers!

To compare the sensitivity of your primers to the sensitivity of those currently used in the Runstadler lab you will examine the C_T values from your RRT-PCR assay. The C_T values are displayed as an amplification curve following RRT-PCR (these values are also given numerically). The initial cycles measure very little fluorescence due to low amounts of double-stranded DNA and are used to establish the inherent background fluorescence. As double-stranded product is produced, fluorescence is measured and the curve appears linear. This linear portion of the curve represents the exponential phase of PCR. Throughout the exponential phase, the curve should be smooth. Sharp points may be due to errors in reaction preparation or failures in the machine used to measure fluorescence. As mentioned previously, the first cycle in which the fluorescence measurement is above background is the C_T. During the later cycles the curve shows minimal increases in fluorescence due the depletion of reagents.

Following the RRT-PCR amplification measurements, a melt curve is completed. Melt curves assess the dissociation of double-stranded DNA while the sample is heated. As the temperature is increased, double-stranded DNA ‘melts’ as the strands dissociate. As discussed above, the fluorescent dye used in RRT-PCR associates with double-stranded DNA and fluorescence measurements will decrease as the temperature increases. In RRT-PCR, the melt curve is used to confirm that a single amplification product was generated during the reaction. If additional products were present, the melt curve would presumably show additional peaks. Why might this be true? Can you think of a scenario where two different products would produce a single peak in a melt curve?

Protocols

Part 1: Prepare primers and setup screen for AIV in bird samples

1. Calculate the amount of water needed for each primer (forward and reverse, separately) to give a concentration of 100 μM.
2. Touch-spin your primers, resuspend each in the appropriate volume of sterile water, vortex, and touch-spin again.
3. Now prepare a dilution from your archival stock. Prepare 100 μL of a solution that has each primer present at 20 μM.
   • Try the calculation on your own first. If you get stuck ask the teaching faculty for help.
   • Be sure to change tips between primers!
4. Return the rest of your primers, plus your primer specification sheets, up front.
5. With your primers you will screen three bird samples for AIV. You will also use the Avian Influenza A Matrix Forward and Reverse primers to screen the samples. Lastly, you will setup 'no template control' reactions with your primer set and the Runstadler Lab primer set.
   • Label 8 microcentrifuge tubes according to the samples you will prepare for RRT-PCR.
   • Obtain the cDNA aliquots from the front lab bench (sample #1, #2, #3).
   • Each reaction should contain 4 μL of cDNA, 2 μL of your diluted primer solution, and water for a total volume of 12.5 μL. For each sample/primer set combination, prepare enough for 2.5 reactions.
   • At the front bench, add the appropriate volume of 2X Power SYBR such that the final concentration in your tubes is 1X. Ask the teaching faculty if you are unsure on how much to add.
6. Add your reactions to the plate on the front bench according to the map. You will add 25 μL to each well.

For your reference, the RRT-PCR conditions will be those shown below:

Stage	Cycles	Details
1	1	95°C for 10 min
2	40	95°C for 15 sec 60°C for 30 sec 72°C for 30 sec
3	1	4°C hold

Part 2: Bird microbiome practice analysis

Part 3: AIV screening analysis

1. Before you examine your data, take a moment to think about what the CT values tell you about sensitivity.
   • Would you expect more sensitive primers to have a higher or lower CT value when compared to less sensitive primers? Why?
2. View the amplification curves and melt curves and melt curves for your RRT-PCR data on the Talk page.
   • Briefly describe the appearance of your amplification curve. Do the samples cross the threshold line during the same cycle? Why or why not?
   • Briefly describe the appearance of your melt curve. Is there a single peak? Why or why not?
3. Go to the Talk page to view the RRT-PCR C_T data collected from your AIV screening assay.
   • Are C_T values available for your ‘no transcript control’ sample? What does this say about the remaining data from your assay?
   • Record the C_T values for the samples you tested with your primers. Are the values similar for the three samples? Can you hypothesize as to which sample(s) were positive and/or negative for the presence of AIV?
   • Record the C_T values for the samples you tested with the Runstadler lab primers. Are the values similar for the three samples? Can you hypothesize as to which sample(s) were positive and/or negative for the presence of AIV?
   • Now, compare the C_T values for your primers and the Runstadler lab primers. Can you make any conclusions concerning the relative

Homework

Due on M1D8

1. Revise the draft of your methods section you submitted on M1D4 applying the feedback you received. In addition, include a write-up of the methods associated with the cloning you completed for this module.

Due on M2D3

1. You will report your findings for the AIV screening portion of this module as a Primer Design Memo. Review the guidelines for this assignment and feel free to get an early start!

Reagent List

• Your brains!

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